Transgenic microbial polyhydroxyalkanoate producers

ABSTRACT

Transgenic microbial strains are provided which contain the genes required for PHA formation integrated on the chromosome. The strains are advantageous in PHA production processes, because (1) no plasmids need to be maintained, generally obviating the required use of antibiotics or other stabilizing pressures, and (2) no plasmid loss occurs, thereby stabilizing the number of gene copies per cell throughout the fermentation process, resulting in homogeneous PHA product formation throughout the production process. Genes are integrated using standard techniques, preferably transposon mutagenesis. In a preferred embodiment wherein multiple genes are incorporated, these are incorporated as an operon. Sequences are used to stabilize mRNA, to induce expression as a function of culture conditions (such as phosphate concentration), temperature, and stress, and to aid in selection, through the incorporation of selection markers such as markers conferring antibiotic resistance.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is application is a continuation of U.S. Ser. No. 11/053,551, filedFeb. 8, 2005, entitled “Transgenic Microbial PolyhydroxyalkanoateProducers” by Gjalt W. Huisman, Oliver P. Peoples, and Frank A. Skraly,which is a continuation of U.S. Ser. No. 10/461,069 filed Jun. 13, 2003,now U.S. Pat. No. 6,913,911, which is a continuation of Ser. No.09/375,975 filed Aug. 17, 1999, now U.S. Pat. No. 6,593,116, whichclaims priority to U.S. provisional application Ser. No. 60/096,852,filed Aug. 18, 1998, the teachings of which are incorporated herein. Thedisclosures in the applications listed above are herein incorporated byreference.

BACKGROUND OF THE INVENTION

The present invention is generally in the field of biosynthesis ofpoly(3-hydroxyalkanoates), and more particularly to improved microbialstrains useful in commercial production of polyhydroxyalkanoates.

Poly(3-hydroxyalkanoates) (PHAs) are biological polyesters synthesizedby a broad range of bacteria. These polymers are biodegradable andbiocompatible thermoplastic materials, produced from renewableresources, with a broad range of industrial and biomedical applications(Williams & Peoples, CHEMTECH 26:38-44 (1996)). PHA biopolymers haveemerged from what was originally considered to be a single homopolymer,poly-3-hydroxybutyrate (PHB) into a broad class of polyesters withdifferent monomer compositions and a wide range of physical properties.About 100 different monomers have been incorporated into the PHApolymers (Steinbuehel & Valentin, FEMS Microbiol. Lett. 128:219-28(1995)).

It has been useful to divide the PHAs into two groups according to thelength of their side chains and their biosynthetic pathways. Those withshort side chains, such as PHB, a homopolymer of R-3-hydroxybutyric acidunits, are crystalline thermoplastics, whereas PHAs with long sidechains are more elastomeric. The former have been known for aboutseventy years (Lemoigne & Roukhelman, 1925), whereas the lattermaterials were discovered relatively recently (deSmet et al., J.Bacteriol. 154:870-78 (1983)). Before this designation, however, PHAs ofmicrobial origin containing both (R)-3-hydroxybutyric acid units andlonger side chain (R)-3-hydroxyacid units from C₅ to C₁₆ had beenidentified (Wallen & Rohweder, Environ. Sci. Technol. 8:576-79 (1974)).A number of bacteria which produce copolymers of (R)-3-hydroxybutyricacid and one or more long side chain hydroxyacid units containing fromfive to sixteen carbon atoms have been identified (Steinbuchel & Wiese,Appl. Microbiol. Biotechnol. 37:691-97 (1992); Valentin et al., Appl.Microbiol. Biotechnol. 36:507-14 (1992); Valentin et al., Appl.Microbiol. Biotechnol. 40:710-16 (1994); Abe et al., Int. J. Biol.Macromol. 16:115-19 (1994); Lee et al., Appl. Microbiol. Biotechnol.42:901-09 (1995); Kato et al., Appl. Microbiol. Biotechnol. 45:363-70(1996); Valentin et al., Appl. Microbiol. Biotechnol. 46:261-67 (1996);U.S. Pat. No. 4,876,331 to Doi). A combination of the two biosyntheticpathways outlined described above provide the hydroxyacid monomers.These copolymers can be referred to as PHB-co-HX (where X is a3-hydroxyalkanoate or alkenoate or alkenoate of 6 or more carbons). Auseful example of specific two-component copolymers isPHB-co-3-hydroxyhexanoate (PHB-co-3HH) (Brandi et al., Int. J. Biol.Macromol. 11:49-55 (1989); Amos & McInerey, Arch. Microbiol. 155:103-06(1991); U.S. Pat. No. 5,292,860 to Shiotani et al.).

PHA production by many of the microorganisms in these references is notcommercially useful because of the complexity of the growth medium, thelengthy fermentation processes, or the difficulty of down-streamprocessing of the particular bacterial strain. Genetically engineeredPHA production systems with fast growing organisms such as Escherichiacoli have been developed. Genetic engineering also allows for theimprovement of wild type PHA production microbes to improve theproduction of specific copolymers or to introduce the capability toproduce different PHA polymers by adding PHA biosynthetic enzymes havingdifferent substrate-specificity or even kinetic properties to thenatural system. Examples of these types of systems are described inSteinbuchel & Valentin, FEMS Microbiol. Lett. 128:219-28 (1995). PCT WO98/04713 describes methods for controlling the molecular weight usinggenetic engineering to control the level of the PHA synthase enzyme.Commercially useful strains, including Alcaligenes eutrophus (renamed asRalstonia eutropha), Alcaligenes latus, Azotobacter vinlandii, andPseudomonads, for producing PHAs are disclosed in Lee, Biotechnology &Bioengineering 49:1-14 (1996) and Braunegg et al., (1998), J.Biotechnology 65: 127-161.

The development of recombinant PHA production strains has followed twoparallel paths. In one case, the strains have been developed to producecopolymers, a number of which have been produced in recombinant E. coli.These copolymers include poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(PHBV), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB-co-4HB),poly(4-hydroxybutyrate) (P4HB) and long side chain PHAs comprising3-hydroxyoctanoate units (Madison and Huisman, 1999. Strains of E. colicontaining the phb genes on a plasmid have been developed to produceP(3HB-3HV) (Slater, et al., Appl. Environ. Microbiol. 58:1089-94 (1992);Fidler & Dennis, FEMS Microbiol Rev. 103:231-36 (1992); Rhie & Dennis,Appl. Environ. Micobiol. 61:2487-92 (1995); Zhang, H. et al., Appl.Environ. Microbiol. 60:1198-205 (1994)). The production of P(4HB) andP(3HB-4HB) in E. coli is achieved by introducing genes from ametabolically unrelated pathway into a P(3HB) producer (Hein, et al.,FEMS Microbiol. Lett. 153:411-18 (1997); Valentin & Dennis, J.Biotechnol. 58:33-38 (1997)). E. coli also has been engineered toproduce medium short chain polyhydroxyalkanoates (msc-PHAs) byintroducing the phaC1 and phaC2 gene of P. aeruginosa in a fadB::kanmutant (Langenbach, et al., FEMS Microbiol. Lett. 150:303-09 (1997); Qi,et al., FEMS Microbiol. Lett. 157:155-62 (1997)).

Although studies demonstrated that expression of the A. eutrophus PHBbiosynthetic genes encoding PHB polymerase, -ketothiolase, andacetoacetyl-CoA reductase in E. coli resulted in the production of PHB(Slater, et al., J. Bacteriol. 170:4431-36 (1988); Peoples & Sinskey, J.Biol. Chem. 264:15298-303 (1989); Schubert, et al., J. Bacteriol.170:5837-47 (1988)), these results were obtained using basic cloningplasmid vectors and the systems are unsuitable for commercial productionsince these strains lacked the ability to accumulate levels equivalentto the natural producers in industrial media.

For commercial production, these strains have to be made suitable forlarge scale fermentation in low cost industrial medium. The first reportof recombinant P(3HB) production experiments in fed-batch cultures usedan expensive complex medium, producing P(3HB) to 90 g/L in 42 hoursusing a pH-stat controlled system (Kim, et al., Biotechnol. Lett.14:811-16 (1992)). Using stabilized plasmids derived from either medium-or high-copy-number plasmids, it was shown that E. coli XL1-Blue withthe latter type plasmid is required for substantial P(3HB) accumulation(Lee, et al., Ann. N.Y. Acad. Sci. 721:43-53 (1994)). In a fed-batchfermentation on 2% glucose/LB medium, this strain produced 81% P(3HB) ata productivity of 2.1 g/L-hr (Lee, et al., J. Biotechnol. 32:203-11(1994)). The P(3HB) productivity was reduced to 0.46 g/L-hr in minimalmedium, but could be recovered by the addition of complex nitrogensources such as yeast extract, tryptone, casamino acids, and collagenhydrolysate (Lee & Chang, Adv. Biochem. Eng. Biotechnol. 52:27-58(1995); Lee, et al., J. Ferment. Bioeng. 79:177-80 (1995)).

Although recombinant E. coli XL1-blue is able to synthesize substantiallevels of P(3HB), growth is impaired by dramatic filamentation of thecells, especially in defined medium (Lee, et al., Biotechnol. Bioeng.44:1337-47 (1994); Lee, Biotechnol. Lett. 16:1247-52 (1994); Wang & Lee,Appl. Environ. Microbiol. 63:4765-69 (1997)). By overexpression of FtsZin this strain, biomass production was improved by 20% and P(3HB) levelswere doubled (Lee & Lee, J. Environ. Polymer Degrad. 4:131-34 (1996)).This recombinant strain produced 104 g/L P(3HB) in defined mediumcorresponding to 70% of the cell dry weight. The volumetric productivityof 2 g/L-hr, however, is lower than achievable with R. eutropha.Furthermore, about 15% of the cells lost their ability to produce PHB bythe end of the fermentation (Wang & Lee, Biotechnol. Bioeng. 58:325-28(1998)).

Recombinant E. coli P(3HB-3HV) producers reportedly are unable to growto a high density and therefore are unsuited for commercial processes(Yim, et al., Biotechnol. Bioeng. 49:495-503 (1996)). In an attempt toimprove P(3HB-3HV) production in a recombinant strain, four E. colistrains (XL1-Blue, JM109, HB101, and DH5α) were tested by Yim et al. Allfour recombinant E. coli strains synthesized P(3HB-3HV) when grown onglucose and propionate with HV fractions of 7% (Yim, et al., Biotechnol.Bioeng. 49:495-503 (1996)). Unlike other strains studied. (Slater, etal., Appl. Environ. Microbiol. 58:1089-94 (1992)), recombinant XL1-Blueincorporates less than 10% HV when the propionic acid concentration isvaried between 0 and 80 mM. HV incorporation and PHA formation wereincreased by pre-growing cells on acetate followed by glucose/propionateaddition at a cell density of around 10⁸ cells per ml. Oleatesupplementation also stimulated HV incorporation. This recombinantXL1-Blue when pregrown on acetate and with oleate supplementationreached a cell density of 8 g/L, 75% of which was P(3HB-3HV) with an HVfraction of 0.16 (Yim, et al., Biotechnol. Bioeng. 49:495-503 (1996)).

One of the challenges of producing P(3HB) in recombinant organisms isthe stable and constant expression of the phb genes during fermentation.Often P(3HB) production by recombinant organisms is hampered by the lossof plasmid from the majority of the bacterial population. Such stabilityproblems may be attributed to the metabolic load exerted by the need toreplicate the plasmid and synthesize P(3HB), which diverts acetyl-CoA toP(3HB) rather than to biomass. In addition, plasmid copy numbers oftendecrease upon continued fermentation because only a few copies providethe required antibiotic resistance or prevent cell death by maintainingparB. For these reasons, a runaway plasmid was designed to suppress thecopy number of the plasmid at 30° C. and induce plasmid replication byshifting the temperature to 38° C. (Kidwell, et al., Appl. Environ.Microbiol. 61:1391-98 (1995)). Using this system, P(3HB) was produced toabout 43% of the cell dry weight within 15 hours after induction with avolumetric production of 1 gram P(3HB) per liter per hour. Although thisproductivity is of the same order of magnitude as natural P(3HB)producers, strains harboring these parB-stabilized runaway repliconsstill lost the capacity to accumulate P(3HB) during prolongedfermentations.

While the instability of the phb genes in high cell-densityfermentations affects the PHA cost by decreasing the cellular P(3HB)yields, the cost of the feedstock also contributes to the comparativelyhigh price of PHAs. The most common substrate used for P(3HB) productionis glucose. Consequently, E. coli and Klebsiella strains have beenexamined for P(3HB) formation on molasses, which cost 33-50% less thanglucose (Zhang, et al., Appl. Environ. Microbiol. 60:1198-1205 (1994)).The main carbon source in molasses is sucrose. Recombinant E. coli andK. aerogenes strains carrying the phb locus on a plasmid grown inminimal medium with 6% sugarcane molasses accumulated P(3HB) toapproximately 3 g/L corresponding to 45% of the cell dry weight. Whenthe K. aerogenes was grown fed-batch in a 10 L fermenter on molasses asthe sole carbon source, P(3HB) was accumulated to 70% its cell dryweight, which corresponded to 24 g/L. Although the phb plasmid in K.aerogenes was unstable, this strain shows promise as a P(3HB) produceron molasses, especially since fadR mutants incorporate 3HV up to 55% inthe presence of propionate (Zhang, et al., Appl. Environ. Microbiol.60:1198-1205 (1994)).

U.S. Pat. No. 5,334,520 to Dennis discloses the production of PHB in E.coli transformed with a plasmid containing the phbCAB genes. A rec⁻,lac⁺ E. coli strain was grown on whey and reportedly accumulates PHB to85% of its cell dry weight. U.S. Pat. No. 5,371,002 to Dennis et al.discloses methods to produce PHA in recombinant E. coli using a highcopy number plasmid vector with phb genes in a host that expresses theacetate genes either by induction, constitutively, or from a plasmid.U.S. Pat. No. 5,512,456 to Dennis discloses a method for production andrecovery of PHB from transformed E. coli strains. These E. coli strainsare equipped with a vector containing the phb genes and a vectorcontaining a lysozyme gene. High copy number plasmids or runawayreplicons are used to improve productivity. The vectors are stabilizedby parB or by supF/dnaB(am). Using such strains, a productivity of 1.7g/L-hr was obtained corresponding to 46 g/L PHB in 25 hrs, after whichthe plasmid was increasingly lost by the microbial population. PCTWO94/21810 discloses the production of PHB in recombinant strains of E.coli and Klebsiella aerogenes with sucrose as a carbon source. PCT WO95/21257 discloses the improved production of PHB in transformedprokaryotic hosts. Improvements in the transcription regulatingsequences and ribosome binding site improve PHB formation by the plasmidbased phb genes. The plasmid is stabilized by the parB locus. PHBproduction by this construct is doubled by including the 361 nucleotidesthat are found upstream of phbC in R. eutropha instead of only 78nucleotides. It is generally believed that PHB production by recombinantmicroorganisms requires high levels of expression using stabilizedplasmids. Since plasmids are available in the cell in multiple copies,ranging from one to several hundreds, the use of plasmids ensured thepresence of multiple copies of the genes of interest. Since plasmids maybe lost, stabilization functions are introduced. Such systems, which aredescribed above, have been tested for PHB production, and the utility ofthese systems in industrial fermentation processes has beeninvestigated. However, overall PHB yield is still affected by loss ofphb genes.

It is therefore an object of the present invention to providerecombinant microorganisms strains useful in industrial fermentationprocesses which can accumulate commercially significant levels of PHBwhile providing stable and constant expression of the phb genes duringfermentation.

It is another object of the present invention to provide transgenicmicrobial strains for enhanced production of poly(3-hydroxyalkanoates).

It is another object of the present invention to provide transgenicmicrobial strains which yield stable and constant expression of the phbgenes during fermentation and accumulate commercially significant levelsof PHB, and methods of use thereof.

SUMMARY OF THE INVENTION

Transgenic microbial strains are provided which contain the genesrequired for PHA formation integrated on the chromosome. The strains areadvantageous in PHA production processes, because (1) no plasmids needto be maintained, generally obviating the required use of antibiotics orother stabilizing pressures, and (2) no plasmid loss occurs, therebystabilizing the number of gene copies per cell throughout thefermentation process, resulting in homogeneous PHA product formationthroughout the production process. Genes are integrated using standardtechniques, preferably transposon mutagenesis. In a preferred embodimentwherein multiple genes are incorporated, these are incorporated as anoperon. Sequences are used to stabilize mRNA, to induce expression as afunction of culture conditions (such as phosphate concentration),temperature, and stress, and to aid in selection, through theincorporation of selection markers such as markers conferring antibioticresistance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are a diagram showing the construction of pMNXTp₁kan,pMNXTp₁cat, pMSXTp₁kan, and pMSXTp₁cat. The inserted sequences are SEQID NO: 1 and SEQ ID NO: 2.

FIGS. 2A and 2B are a diagram showing the construction of pMUXC₅cat.

FIGS. 3A-3C are a diagram showing the construction of pMUXAB₅cat,pMUXTp₁ AB₅kan, pMUXTp₁₁AB₅kan, pMUXTp₁₂AB₅kan, and pMUXTp₁₃AB₅kan (SEQID NO: 3-14).

DETAILED DESCRIPTION OF THE INVENTION

By randomly inserting genes that encode PHA biosynthetic enzymes intothe chromosome of E. coli, means have been identified to directlyachieve high levels of expression from strong endogenous promoters atsites that are non-essential for growth of the host in industrial mediumbased fermentations. As demonstrated by the examples, E. coli strainshave been obtained using these techniques that produce PHAs in levelsexceeding 85% of the cell dry weight from single copy genes on thechromosome. Expression of the phb genes in these strains is notdependent on the upstream sequences of phbC in R. eutropha nor on a highcopy number construct. Maintenance of the phb genes by these strains isindependent of the supplementation of antibiotics, the presence ofstabilizing loci such as parB or hok/sok or any other selectivepressure. The ultra-high level of expression required in theplasmid-based systems has been found to be completely unnecessary.Furthermore, unlike the most successful fermentations reported to date(Wang & Lee, Biotechnol. Bioeng. 58:325-28 (1998)) for recombinantplasmid-based E. coli, fermentation with these strains provides thatvirtually all of the cells contain PHB at the end of the fermentation.

Despite the low copy number, these transgenic bacteria accumulate PHB tolevels observed for wild-type organisms. The host used for recombinantPHB production also is an important parameter in designing aplasmid-based E. coli system. For example, although W3110 strains werepoor PHB producers when using a plasmid-based system, it was found thatby integrating the phb genes into the chromosome of this same host, thehost retained excellent growth characteristics while accumulatingcommercially significant levels of PHB.

Methods and Materials for Producing the Microbial Strains

Bacterial Strains to be Modified

A number of bacteria can be genetically engineered to producepolyhydroxyalkanoates. These include organisms that already producepolyhydroxyalkanoates, modified to utilize alternative substrates orincorporate additional monomers, or to increase production, andorganisms that do not produce polyhydroxyalkanoates, but which expressesnone to some of the enzymes required for production ofpolyhydroxylkanoates. Examples include E. coli, Alcaligenes latus,Alcaligenese eutrophus, Azotobacter, Pseudomonas putida, and Ralstoniaeutropha.

Methods for Generating Transgenic PHB Producers

Methods for incorporating engineered gene constructs into thechromosomal DNA of bacterial cells are well known to those skilled inthe art. Typical integration mechanisms include homologous recombinationusing linearized DNA in recBC or recD strains followed by P1transduction (Miller 1992, A Short Course in Bacterial Genetics: Alaboratory manual & Handbook for Escherichia coli and Related Bacteria.Cold Spring Harbor laboratory Press, Cold Spring Harbor, N.Y.) specialplasmids (Hamilton et al., J. Bacteria 171:4617 (1989); Metcalf et al.,Plasmid 35:1 (1996); U.S. Pat. No. 5,470,727 to Mascarenhas et al.), orby random insertion using transposon based systems (Herrero et al. J.Bacteriol. 172:6557 (1990); Peredelchuk & Bennett, Gene 187:231 (1997);U.S. Pat. No. 5,595,889 to Richaud et al.; U.S. Pat. No. 5,102,797 toTucker et al.). In general, the microbial strains containing aninsertion are selected on the basis of an acquired antibiotic resistancegene that is supplied by the integrated construct. However,complementation of auxotrophic mutants can also be used.

Expression of the genes of interest for chromosomal integration can beachieved by including a transcription activating sequence (promoter) inthe DNA construct to be integrated. Site-directed, homologousrecombination can be combined with amplification of expression of thegenes of interest, as described by U.S. Pat. No. 5,00,000 to Ingram etal. Although mini-transposon systems have been used for a number ofyears, they have been designed such that the expression level of theintegrated gene of interest is not modulated. Ingram, et al. selectedfor increased expression of a foreign gene inserted into the E. colichromosome by homologous recombination. This was achieved by inserting apromoter-less chloroamphenicol (Cm) resistance gene downstream of thegene of interest to create a transcriptional fusion. After atranscriptional fusion of the alcohol dehydrogenase gene with apromoterless chloramphenicol acetyl transferase genes is integrated inthe pfl gene, increased expression is achieved by selecting mutants onincreasing concentrations of chloramphenicol. However, in chemostatstudies these stabilized strains still lost the capacity to produceethanol (Lawford & Rousseau, Appl. Biochem. Biotechnol. 57-58:293-305(1996)). Also, strains that contained the ethanologenic genes on thechromosome demonstrated a decreased growth rate in glucose minimalmedium (Lawford & Rousseau, Appl. Biochem. Biotechnol., 57-58:277-92(1996)).

These approaches have been combined and modified to randomly integrate amini-transposon into the chromosome to select for healthy, fast growingtransgenic strains coupled with a screening system for modulatingexpression of the integrated genes. A series of expression cassetteshave been developed for inserting heterologous genes into bacterialchromosomes. These cassettes are based on the transposon deliverysystems described by Herrero et al., J. Bacteriol. 172:6557-67 (1990);de Lorenzo et al., J. Bacteriol. 172:6568 (1990). Although these systemsspecify RP4-mediated conjugal transfer and use only transposon Tn10 andTn5, any combination of transposon ends and delivery system could beadapted for the technology described, resulting in sustained andhomogeneous PHA production.

The following general approach is used for generating transgenic E. coliPHB producers: (1) a promoterless antibiotic resistance (abr) gene iscloned in the polylinker of a suitable plasmid such as pUC18NotI orpUC18SfiI so that the major part of the polylinker is upstream of abr;(2) phb genes are subsequently cloned upstream of and in the sameorientation as the abr gene; (3) the phb-abr cassette is excised as aNotI or AvrII fragment (AvrII recognizes the SfiI site in pUC18SfiI) andcloned in the corresponding sites of any plasmid like those from thepUT- or pLOF-series; (4) the resulting plasmids are maintained in E.coli λpir strains and electroporated or conjugated into the E. colistrain of choice in which these plasmids do not replicate; and (5) newstrains in which the phb-abr cassette has successfully integrated in thechromosome are selected on selective medium for the host (e.g.,naladixic acid when the host is naladixic acid resistant) and for thecassette (e.g., chloramphenicol, kanamycin, tetracyclin, mercurychloride, bialaphos). The resulting phb integrants are screened onminimal medium in the presence of glucose for growth and PHB formation.

Several modifications of this procedure can be made. If the promotorlessantibiotic resistance marker is not used, the insertion of the PHA genesis selected based on a marker present in the vector and integratedstrains producing the desired level of PHA are detected by screening forPHA production. The phb genes may have, but do not need, endogeneoustranscription sequences, such as upstream activating sequences, RNApolymerase binding site, and/or operator sequences. If the phb genes donot have such sequences, the described approach is limited to the use ofvectors like the pUT series in which transcription can proceed throughthe insertion sequences. This limitation is due to the inability of RNApolymerase to read through the Tn10 flanking regions of the pLOFplasmids. The abr gene may carry its own expression sequences if sodesired. Instead of an abr gene, the construct may be designed such thatan essential gene serves as selective marker when the host strain has amutation in the corresponding wild-type gene. Examples of genes usefulfor this purpose are generally known in the art. Different constructscan be integrated into one host, either subsequently or simultaneously,as long as both constructs carry different marker genes. Using multipleintegration events, phb genes can be integrated separately, e.g., thePHB polymerase gene is integrated first as a phbC-cat cassette, followedby integration of the thiolase and reductase genes as a phbAB-kancassette. Alternatively, one cassette may contain all phb genes whereasanother cassette contains only some phb genes required to produce adesired PHA polymer.

In some cases a transposon integration vector such as pJMS11 (Panke etal. Appl. Enviro. Microbiol. 64: 748-751) may be used such that theselectable marker can be excised from the chromosome of the integratedstrain. This is useful for a number of reasons including providing amechanism to insert multiple transposon constructs using the same markergene by excising the marker following each insertion event.

Sources of phb and Other Genes Involved in PHA Formation

A general reference is Madison and Huisman, 1999, Microbiology andMolecular Biology Reviews 63: 21-53. The phb genes may be derived fromdifferent sources and combined in a single organism, or from the samesource.

Thiolase Encoding Genes

Thiolase encoding genes have been isolated from Alcaligenes latus,Ralstonia eutropha (Peoples & Sinskey, J. Biol. Chem. 264(26):15298-303(1989); Acinetobacter sp. (Schembri, et al., J. Bacteriol.177(15):4501-7 (1995)), Chromotium vinosum (Liebergesell & Steinbuchel,Eur. J. Biochem. 209(1):135-50 (1992)), Pseudomonas acidophila,Pseudomonas denitrificans (Yabutani, et al., FEMS Microbiol. Lett. 133(1-2):85-90 (1995)), Rhizobium meliloti (Tombolini, et al., Microbiology141:2553-59 (1995)), Thiocystis violacea (Liebergesell & Steinbuchel,Appl. Microbiol. Biotechnol. 38(4):493-501 (1993)), and Zoogloearamigera (Peoples, et al., J. Biol. Chem. 262(1):97-102 (1987)).

Other genes that have not been implicated in PHA formation but whichshare significant homology with the phb genes and/or the correspondinggene products may be used as well. Genes encoding thiolase- andreductase-like enzymes have been identified in a broad range of non-PHBproducing bacteria. E. coli (U29581, D90851, D90777), Haemophilusinfluenzae (U32761), Pseudomonas fragi (D10390), Pseudomonas aeruginosa(U88653), Clostridium acetobutylicum (U08465), Mycobacterium leprae(U00014), Mycobacterium tuberculosis (Z73902), Helicobacter pylori(AE000582), Thermoanaerobacterium thermosaccharolyticum (Z92974),Archaeoglobus fulgidus (AE001021), Fusobacterium nucleatum (U37723),Acinetobacter calcoaceticus (L05770), Bacillus subtilis (D84432, Z99120,U29084), and Synechocystis sp. (D90910) all encode one or more thiolasesfrom their chromosome. Eukaryotic organisms such as Saccharomycescerevisiae (L20428), Schizosaccharomyces pombe (D89184), Candidatropicalis (D13470), Caenorhabditis elegans (U41105), human (S70154),rat (D13921), mouse (M35797), radish (X78116), pumpkin (D70895), andcucumber (X67696) also express proteins with significant homology to the3-ketothiolase from R. eutropha.

Reductase Encoding Genes

Reductase encoding genes have been isolated from A. latus, R. eutropha(Peoples & Sinskey, J. Biol. Chem. 264(26):15298-303 (1989);Acinetobacter sp. (Schembri, et al., J. Bacteriol. 177(15):4501-7(1995)), C. vinosum (Liebergesell & Steinbuchel, Eur. J. Biochem.209(1):135-50 (1992)), P. acidophila, P. denitrificans (Yabutani, etal., FEMS Microbiol. Lett. 133 (1-2):85-90 (1995)), R. meliloti(Tombolini, et al., Microbiology 141:2553-59 (1995)), and Z. ramigera(Peoples, et al., J. Biol. Chem. 262(1):97-102 (1987)).

Other genes that have not been implicated in PHA formation but whichshare significant homology with the phb genes and/or the correspondinggene products may be used as well. Genes with significant homology tothe phbB gene encoding acetoacetyl CoA reductase have been isolated fromseveral organisms, including Azospirillum brasiliense (X64772, X52913)Rhizobium sp. (U53327, Y00604), E. coli (D90745), Vibrio harveyi(U39441), H. influenzae (U32701), B. subtilis (U59433), P. aeruginosa(U91631), Synechocystis sp. (D90907), H. pylori (AE000570), Arabidopsisthaliana (X64464), Cuphea lanceolata (X64566) and Mycobacteriumsmegmatis (U66800).

PHA Polymerase Encoding Genes

PHA polymerase encoding genes have been isolated from Aeromonas caviae(Fukui & Doi, J. Bacteriol. 179(15):4821-30 (1997)), A. latus, R.eutropha (Peoples & Sinskey, J. Biol. Chem. 264(26):15298-303 (1989);Acinetobacter (Schembri, et al., J. Bacteriol. 177(15):4501-7 (1995)),C. vinosum (Liebergesell & Steinbuchel, Eur. J. Biochem. 209(1):135-50(1992)), Methylobacterium extorquens (Valentin & Steinbuchel, Appl.Microbiol. Biotechnol. 39(3):309-17 (1993)), Nocardia corallina (GenBankAcc. No. AF019964), Nocardia salmonicolor, P. acidophila, P.denitrificans (Ueda, et al., J. Bacteriol. 178(3):774-79 (1996)),Pseudomonas aeruginosa (Timm & Steinbuchel, Eur. J. Biochem.209(1):15-30 (1992)), Pseudomonas oleovorans (Huisman, et al., J. Biol.Chem. 266:2191-98 (1991)), Rhizobium etli (Cevallos, et al., J.Bacteriol. 178(6):1646-54 (1996)), R. meliloti (Tombolini, et al.,Microbiology 141 (Pt 10):2553-59 (1995)), Rhodococcus ruber (Pieper &Steinbuchel, FEMS Microbiol. Lett. 96(1):73-80 (1992)), Rhodospirrilumrubrum (Hustede, et al., FEMS Microbiol. Lett. 93:285-90 (1992)),Rhodobacter sphaeroides (Steinbuchel, et al., FEMS Microbiol. Rev.9(2-4):217-30 (1992); Hustede, et al., Biotechnol. Lett. 15:709-14(1993)), Synechocystis sp. (Kaneko, DNA Res. 3(3):109-36 (1996)), T.violaceae (Liebergesell & Steinbuchel, Appl. Microbiol. Biotechnol.38(4):493-501 (1993)), and Z. ramigera (GenBank Acc. No. U66242).

Vectors for Incorporation of Genes into the Bacterial Chromosomes

The pUT and pLOF series of plasmid transposon delivery vectors useful inthe PHA-producing methods described herein use the characteristics oftransposon Tn5 and transposon Tn10, respectively. The transposase genesencoding the enzymes that facilitate transposition are positionedoutside of the ‘transposase recognition sequences’ and are consequentlylost upon transposition. Both Tn5 and Tn10 are known to integraterandomly in the target genome, unlike, for example, the Tn7 transposon.However, generally any transposon can be modified to facilitate theinsertion of heterologous genes, such as the phb genes, into bacterialgenomes. This methodology thus is not restricted to the vectors used inthe methods described herein.

Methods and Materials for Screening for Enhanced Polymer Production

Screening of Bacterial Strains

The technology described above allows for the generation of new PHAproducing strains and also provides new bacterial strains that areuseful for screening purposes. Table 1 below shows the differentcombinations of chromosomally and plasmid encoded PHB enzymes and howspecific strains can be used to identify new or improved enzymes.

Besides a screening tool for genes that express improved enzymes, E.coli strains with a complete PHA pathway integrated on the chromosomecan be used to screen for heterologous genes that affect PHA formation.E. coli is a useful host because genes are easily expressed from amultitude of plasmid vectors: high copy-number, low copy-number,chemical or heat inducible, etc. and mutagenesis procedures have beenwell established for this bacterium. In addition, the completelydetermined genomic sequence of E. coli facilitates the characterizationof genes that affect PHA metabolism.

Transgenic E. coli strains expressing an incomplete PHA pathway can betransformed with gene libraries to identify homologs of the missing genefrom other organisms, either prokaryotic or eukaryotic. Because thesescreening strains do not have the complete PHA biosynthetic pathway, themissing functions can be complemented and identified by the ability ofthe host strain to synthesize PHA. Generally PHA synthesizing bacterialcolonies are opaque on agar plates, whereas colonies that do notsynthesize PHA appear translucent. Clones from a gene library thatcomplement the missing gene confer a white phenotype to the host whengrown on screening media. Generally screening media contains allessential nutrients with excess carbon source and an antibiotic forwhich resistance is specified by the vector used in the libraryconstruction.

Besides new genes, genes encoding improved PHA biosynthetic enzymes canalso be screened for. A mutagenized collection of plasmids containing aphb biosynthetic gene into an E. coli host strain lacking this activitybut containing genes encoding the other PHA biosynthetic enzymes can bescreened for increased or altered activity. For example, PHA polymeraseswith increased activity can be screened for in a strain that expressesthiolase and reductase from the chromosome by identifying PHB-containingcolonies under conditions that support PHB formation poorly. mcl-PHApolymerases with an increased specificity towards C₄ can similarly bescreened for under PHB accumulation promoting conditions. Alteredactivities in the phaG encoded ACP::CoA transferase can be screened forby expressing mutated versions of this gene in a phbC integrant andscreening for PHB formation from short chain fatty acids. Enzymes thathave increased activity under sub-optimal physical conditions (e.g.,temperature, pH, osmolarity, and oxygen tension) can be screened for bygrowing the host under such conditions and supplying a collection ofmutated versions of the desired gene on a plasmid. Reductase enzymeswith specificity to medium side-chain 3-ketoacyl-CoA's, such as3-ketohexanoyl-CoA, can be screened for by identifying PHA synthesizingcolonies in a strain that has a msc-PHA polymerase gene integrated onthe chromosome and mutagenized versions of a phbB gene on a plasmid. Thecombination of different specificity PHA enzymes allows for thescreening of a multitude of new substrate specificities. Furtherpermutations of growth conditions allows for screening of enzymes activeunder sub-optimal conditions or enzymes that are less inhibited bycellular cofactors, such as Coenzyme A and CoA-derivatives, reduced oroxidised nicotinamide adenine dinucleotide or nicotinamide adeninedinucleotide phosphate (NAD, NADP, NADH, and NADPH).

Using the techniques described herein, E. coli strains expressing thegenes encoding enzymes for the medium side-chain PHA pathway can beconstructed. Strains in which either phaC or phaG or both are integratedon the chromosome of E. coli accumulate a PHA including mediumchain-length 3-hydroxy fatty acids of which 3-hydroxydecanoate is thepredominant constituent. When phaC is integrated by itself, msc-PHAs canbe synthesized from fatty acids. In such strains, it is advantageous tomanipulate fatty acid oxidation such that 3-hydroxy fatty acidprecursors accumulate intracellularly. This manipulation can be achievedby mutagenesis or by substituting the E. coli fatty acid degradationenzymes FadA and FadB encoding genes with the corresponding faoAB genesfrom Pseudomonas putida or related rRNA homology group I fluorescentpseudomonad.

TABLE 1 Phenotypes of Strains for Screening of New or Improved EnzymesGenes integrated Gene(s) on Carbon source Screen identifies onchromosome plasmid for screen genes encoding phbC library glucose newthiolase/ reductase library fatty acids new reductase, hydratase,transferase library hydroxy fatty acid, hydroxy fatty acid e.g.4-hydroxybutyrate activating enzyme, (4HB) e.g. 4HB-CoA transferase(acetyl- CoA or succinyl-CoA dependent) or 4HB- CoA synthase phaGglucose transferase with new substrate specificity phbAB library glucosenew polymerase gene phaC glucose; altered polymerase with newenvironmental substrate specificity; conditions increased activity undersub-optimal conditions phbBC library glucose new thiolase phbA limitingglucose/less deregulated thiolase; prefered carbon increased activitysources or rich under sub-optimal medium; altered conditionsenvironmental conditions phbAC library glucose new reductase phbBlimiting glucose/less deregulated reductase; prefered carbon increasedactivity sources or rich under sub-optimal medium; altered conditionsenvironmental conditions phbCAB library any enzymes affecting PHBformation under specific conditions phbCAB, random any enzymes affectingmutations PHB formation under (chemical or specific conditionstransposon) phaC library hexanoate hydratase with specificity for C6 andlonger substrates phaJ fatty acids hydratase with increased specificityfor C6 and longer substrates phbB fatty acids reductase with newsubstrate specificity phaC fadR⁺, Δato phbAB glucose + butyratetbiolase/reductase combination specific for C6 monomer phaJ phaC fattyacids polymerase with wider substrate specificity phaG phbC glucosepolymerase with wider substrate specificity

EXAMPLES

The methods and compositions described herein will be further understoodby reference to the following non-limiting examples. These examples usethe following general methods and materials.

Materials and Methods

E. coli strains were grown in Luria-Bertani medium. (Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 2d ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. [1992 at 37° C. or 30° C. orin minimal E2 medium (Lageveen et al., Appl. Environ. Microbiol. 54:2924-2932 (1988)). DNA manipulations were performed on plasmid andchromosomal DNA purified with the Qiagen plasmid preparation or Qiagenchromosomal DNA preparation kits according to manufacturersrecommendations. DNA was digested using restriction enzymes (New EnglandBiolabs, Beverly, Mass.) according to manufacturers recommendations. DNAfragments were isolated from 0.7% agarose-Tris/acetate/EDTA gels using aQiagen kit.

Plasmid DNA was introduced into E. coli cells by transformation orelectroporation (Sambrook, et al., Molecular Cloning: A LaboratoryManual, 2d ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.)). Transposition of phb genes from the pUT vectors was achieved bymating of the plasmid donor strain and the recipient (Herrero et al., J.Bacteriol. 172:6557 (1990)). The recipient strains used were spontaneousnaladixic acid or rifampicin resistant mutants of E. coli derived fromeither LS5218 or MBX23. MBX23 is LJ14 rpoS::Tn10 in which the rpoS::Tn10allele was introduced by P1 transduction from strain 1106 (Eisenstark).Recipients in which phb genes have been integrated into the chromosomewere selected on naladixic acid or rifampicin plates supplemented withthe antibiotic resistance specified by the mini-transposon, kanamycin,or chloramphenicol. Oligonucleotides were purchased from Biosynthesis orGenesys. DNA sequences were determined by automated sequencing using aPerkin-Elmer ABI 373A sequencing machine. DNA was amplified using thepolymerase-chain-reaction in 50 microliter volume using PCR-mix fromGibco-BRL (Gaithersburg, Md.) and an Ericomp DNA amplifying machine.

DNA fragments were separated on 0.7% agarose/TAE gels. Southern blotswere performed according to procedures described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 2d ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). Detection of DNA fragmentscontaining phb genes was performed using chemiluminescent labeling anddetection kits from USB/Amersham. Proteins samples were denatured byincubation in a boiling water bath for 3 minutes in the presence of2-mercaptoethanol and sodium dodecylsulphate and subsequently separatedon 10%, 15%, or 10-20% sodium dodecylsulphate-polyacrylamide gels. Aftertransfer of protein to supported nitrocellulose membranes (Gibco-BRL,Gaithersburg, Md.), 3-ketoacyl-CoA thiolase, acetoacetyl-CoA reductaseand PHB polymerase was detected using polyclonal antibodies raisedagainst these enzymes and horseradish peroxidase labeled secondaryantibodies followed by chemiluminescent detection (USB/Amersham).

Acetoacetyl-CoA thiolase and acetoacetyl-CoA reductase activities weredetermined as described by Peoples and Sinskey, J. Biol. Chem. 264:15293-15297 (1989) in cell free extracts from strains grown for 16 hoursin LB-medium at 37° C. The acetoacetyl-CoA thiolase activity is measuredas degradation of a Mg²⁺-acetoacetyl-CoA complex by monitoring thedecrease in absorbance at 304 nm after addition of cell-free extractusing a Hewlett-Packer spectrophotometer. The acetoacetyl-CoA reductaseactivity is measured by monitoring the conversion of NADH to NAD at 340nm using a Hewlett-Packer spectrophotometer.

Accumulated PHA was determined by gas chromatographic (GC) analysis asfollows. About 20 mg of lyophilized cell mass was subjected tosimultaneous extraction and butanolysis at 110° C. for 3 hours in 2 mLof a mixture containing, by volume, 90% 1-butanol and 10% concentratedhydrochloric acid, with 2 mg/mL benzoic acid added as an internalstandard. The water-soluble components of the resulting mixture wereremoved by extraction with 3 mL water. The organic phase (1 μL at asplit ratio of 1:50 at an overall flow rate of 2 mL/min) was analyzed onan HP 5890 GC with FID detector (Hewlett-Packard Co, Palo Alto, Calif.)using an SPB-1 fused silica capillary GC column (30 m; 0.32 mm ID; 0.25μm film; Supelco; Bellefonte, Pa.) with the following temperatureprofile: 80° C., 2 min.; 10° C. per min. to 250° C.; 250° C., 2 min. Thestandard used to test for the presence of 4-hydroxybutyrate units in thepolymer was γ-butyrolactone, which, like poly(4-hydroxybutyrate), formsn-butyl 4-hydroxybutyrate upon butanolysis. The standard used to testfor 3-hydroxybutyrate units in the polymer was purified PHB.

The molecular weights of the polymers were determined followingchloroform extraction by gel permeation chromatography (GPC) using aWaters Styragel HT6E column (Millipore Corp., Waters ChromatographyDivision, Milford, Mass.) calibrated versus polystyrene samples ofnarrow polydispersity. Samples were dissolved in chloroform at 1 mg/mL,and 50 μL samples were injected and eluted at 1 mL/min. Detection wasperformed using a differential refractometer.

1-Methyl-3-nitro-1-nitroso-guanidine (NTG) mutagenesis was performed asdescribed by Miller, A Short Course in Bacterial Genetics (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.) using a 90 minutetreatment with 1 mg/ml NTG corresponding to 99% killing.

Example 1 Host Strains and Plasmid Tools for Gene Integration

Strains and plasmids from which transposon vectors and transposonderivatives were developed are listed in Tables 2 and 3 below. MBX245and MBX247 were selected by growing MBX23 and LS5218 respectively on LBplates containing approximately 30 μg/ml naladixic acid. MBX246 andMBX248 were selected by growing MBX23 and LS5218, respectively, on LBplates containing 50 μg/ml rifampicin. Colonies that appeared on theseselective media within 24 hours were replica plated on the same mediumand after growth stored in 15% glycerol/nutrient broth at −80° C.

MBX245 and MBX247 were selected by growing MBX23 and LS5218 respectivelyon LB plates containing 30 μg/ml naladixic acid. MBX246 and MBX248 wereselected by growing MBX23 and LS5218 respectively on LB platescontaining 50 μg/ml rifampicin. Colonies that appeared on theseselective media within 24 hours were replica plated on the same mediumand after growth stored in 15% glycerol/nutrient broth at −80° C.

TABLE 2 Host Strains Used For Gene Integration strain genotype sourceDH5α recA1 endA1 gyrA96 thi 1 hsdR17 supE44 relA1 Δ(lac- proAB)(Φ80dlacΔ(lacZ)M15) S17-1 λpir recA thi pro hsdR⁻M⁺ RP4:2- 2 Tc::Mu::KmTn7 λpir lysogen CC118 λpir Δ(ara-leu) araD ΔlacX74 galE 2 galK phoA20thi-1 rpsE rpoB argE(Am) recA1, λ pir lysogen XL1-Blue F′::Tn10 lacI^(q)Δ(lacZ) M15 3 proAB/recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 Δ(lac-proAB) LS5218 fadR601 atoC512^(c) Spratt et al, 1981 J. Bacteriol. 146:1166-1169 LJ14 LS5218 atoC2^(c) atoA14 Spratt et al, 1981 J. Bacteriol.146: 1166-1169 MBX23 LJ14 rpoS Metabolix, Inc. MBX245 MBX23 Nl^(r)Metabolix, Inc. MBX246 MBX23 Rf^(r) Metabolix, Inc. MBX247 LS5218 Nl^(r)Metabolix, Inc. MBX248 LS5218 Rf^(r) Metabolix, Inc. 1. New EnglandBiolabs (Beverly, MA) 2. Herrero et al., J. Bacteriol. 172: 6557-67(1990) 3. Stratagene (San Diego, CA)

TABLE 3 Plasmids Used For Gene Integration plasmid characteristicssource pUC18Not Ap^(r), NotI sites flanking polylinker 2 pUC18SfiAp^(r), SfiI sites flanking polylinker 2 pUTkan Ap^(r), Km^(r), oriR6K,mobRP4 depends 2 on λpir for replication pUTHg Ap^(r), Hg^(r), oriR6K,mobRP4 depends 2 on λpir for replication pKPS4 Ap^(r), phaC1 fromPseudomonas oleovorans pUCDBK1 Ap^(r), phbA and phbB from ZoogloeaPeoples and ramigera Sinskey 1989, Molecular Microbiol. 3: 349-357 pZSAp^(r), phbC from Zoogloea ramigera WO 99/14313

Example 2 Construction of Cloning Vectors to Facilitate Integration ofphb Genes

The plasmids pMNXTp1kan and pMNXTp1cat were based on the plasmidspUC18Not and pUC18Sfi and developed as shown in FIGS. 1A-1C.

The Tn903 kanamycin (Km) resistance gene from plasmid pBGS18 wasamplified by PCR using the oligonucleotide primers linkK1,

5′ TGCATGCGATATCAATTGTCCA GCCAGAAAGTGAGG (SEQ ID NO: 15),

and linkK2, 5′ ATTTATTCAACAAAGCCGCC (SEQ ID NO: 16).

Prior to PCR amplification, the primers were phosphorylated using T4polynucleotide kinase using standard procedures. The DNa was amplifiedusing the following program: 1 cycle of 3 min at 95° C., 40 s at 42° C.,2 min at 72° C., followed by 30 cycles of 40 s at 95° C., 40 s at 42° C.and 90 s at 72° C. The DNA then was phenol extracted and treated with T4DNA polymerase prior to gel purification. The blunt ended 0.8 kb DNAfragment was then inserted into the Ecl136II site in the polylinker ofpUC18Not to obtain pMNXkan.

The cat gene was obtained as an HindIII cassette from Pharmacia(Pharmacia Inc. NJ), blunt ended using Klenow fragment of DNApolymerase, and inserted into the Ecl136II site of pUC18Not to obtainpMNXcat.

The trp terminator sequence was constructed by annealing the twosynthetic oligonucleotides TERM1

(5′ CCCAGCCCGCTAATGAGCGGGCTTTTTTTTGAACAA AA 3′) (SEQ ID NO: 17) andTERM2 (5′ TACGTATTTTGTTCAAAAAAAAGCCCGCTCATTAGCGGG CTGGG 3′) (SEQ ID NO:18).

The terminator was then inserted into the HindIII-SphI site of pMNXkanand pMNXcat to obtain pMNXTkan and pMNXTcat, respectively. These vectorswere constructed such that any promoter fragment can be added betweenthe SphI and SacI sites. Promoter p₁ was constructed by annealing of thesynthetic oligonucleotides PHBB1

(5′ TACGTACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGT GTGGAATTG TGAGCGGTT3′) (SEQ ID NO: 19) and PHBB2

(5′ TTCGAACCGCTCACAATTCCACACAACATACGAGCCGGAAGC ATAAATGTAAAGCCTGGGG 3′)(SEQ ID NO: 20), followed by filling in the ends with Klenow fragment ofDNA polymerase. The blunt-ended promoter fragment p₁ was then insertedinto the HincII site of pMNXTkan and pMNXTcat to obtain pMNXTp₁kan andpMNXTp₁cat, respectively.

Plasmid pMSXTp₁cat was constructed by transferring the Tp₁cat cassettefrom pMNXTp₁cat as an EcoRI-HindIII fragment into the EcoRI-HindIII siteof pUC18Sfi. Similarly, pMSXTp₁kan was constructed by transferring theEcoRI-HindIII fragment containing Tp₁kan into the EcoRI-HindIII site ofpUC18Sfi.

Example 3 Construction of Plasmids for Chromosomal Integration of phbC,Encoding PHB polymerase

Plasmid pMUXC₅cat contains the phbC gene from Z. ramigera on atransposable element for integration of this gene on the chromosome of arecipient strain, as shown in FIGS. 2A and 2B. Strong translationalsequences were obtained from pKPS4 which includes phaC1 encoding PHApolymerase from P. oleovorans in the pTrc vector (Pharmacia). In thisconstruct, phaC1 is preceded by a strong ribosome binding site:AGGAGGTTTTT(-ATG) (SEQ ID NO: 21). The phaC1 gene including the upstreamsequences, was cloned as a blunt ended EcoRI-HindIII fragment in theSmaI site of pUC18Sfi to give pMSXC₃. A blunt ended cat gene cassettewas subsequently cloned in the blunt-ended Sse8387II site, resulting inpMSXC₃cat. At this point, all of the phaC1 coding region except the 5′27 base pairs were removed as a PstI-BamHI fragment and replaced by thecorresponding fragment from the phbC gene from Z. ramigera. Theresulting plasmid pMSXC₅cat encodes a hybrid PHB polymerase enzyme withthe 9 amino terminal residues derived from the P. oleovorans PHApolymerase and the remainder from Z. ramigera. The C₅cat cassette wasthen excised as an AvrII fragment and cloned in the corresponding sitesof pUTHg, thereby deleting the mercury resistance marker from thisvector. The resulting plasmid, pMUXC₅cat, contains a C₅catmini-transposon in which phbC is not preceded by a promoter sequence.Expression of the cassette upon integration is therefore dependent ontranscriptional sequences that are provided by the DNA adjacent to theintegration site.

Example 4 Construction of Plasmids for Chromosomal Integration of phbAB,Encoding Thiolase and Reductase

pMSXTp₁AB₅kan2 was constructed from pMSXTp₁kan as partially shown inFIGS. 3A-3C. First pMSXTp₁kan was digested with NdeI, filled in withKlenow and religated to obtain pMSXTp₁kan2 in which the NdeI site isdeleted. This deletion results in a unique NdeI site just upstream ofphbA of Z. ramigera during later stages of the cloning procedure.

B₅ was cloned as a NarI fragment from pUCDBK1 (Peoples and Sinskey 1989,Molecular Microbiol. 3: 349-357) and cloned in the HincII site ofpUC18Sfi to generate pMSXB₅. A₅ was inserted as an FseI/blunt-SalIfragment in the Ecl136II-SalI sites resulting in pMSXAB₅ andregenerating the Z. ramigera AB₅ intergenic region. pMSXAB₅cat wascreated by inserting a promoterless cat cassette in the HindIII site ofpMSXAB₅. The AB₅ fragment from pMSXAB₅cat was cloned as a EcoRI-PstIfragment into the SmaI site of pMSXTp₁kan2 giving pMSXTp₁AB₅kan2.

The expression cassette AB₅cat was then excised as a 2.8 kb AvrIIfragment and ligated into the AvrII site of pUTHg and transformed intoE. coli strain CC118 λpir to obtain plasmid pMUXAB₅cat. This plasmid wasthen transformed into E. coli S17-1λpir and used to insert the AB5catexpression cassette into the chromosome of E. coli MBX247 byconjugation. The resulting Ap^(s)/Cm^(r) transconjugants werecharacterized for integration and expression of the thiolase andreductase genes encoded by the phbAB genes.

Example 5 Construction of Plasmids with Improved Promoters forIntegration of phbAB into the Chromosome of E. coli

Expression of phbAB5 was improved by introduction of strong promotersupstream of these genes, as shown in FIGS. 3A-3C. These promoters weregenerated with sets of oligonucleotides that provide upstream activatingsequences, a −35 promoter region, a −10 promoter region withtranscriptional start site(s), and mRNA sequences with possiblestabilizing functions. Plasmid pMSXTp₁AB₅kan2 was digested withPstI/XbaI, and a fragment containing the −10 region of the lac promoterwas inserted as a fragment obtained after annealing oligonucleotides 3A

(5′ GGCTCGTATAATGTGTGGAGGGAGAACCGCCGGGCTCGCGCCGTT) (SEQ ID NO: 5) and 3B(5′ CTAGAACGGCGCGAGCCCGGCGGTTCTCCCTCCACACATTATAC GAGCCTGCA) (SEQ ID NO:6).

Next, a fragment containing the lac-35 region and the rrnB region wereinserted into the PstI site as a fragment obtained after annealing theoligonucleotides: 1A

(5′ TTCAGAAAATTATTTTAAATTTCCTCTTGACATTTATGCT GCA) (SEQ ID NO: 7) and 1B

(5′ GCATAAATGTCAAGAGGAAATTTAAAATAATTTTCTGAATGCA) (SEQ ID NO: 8). Next,the messenger stabilizing sequence including the transcriptional startsite from AB₅ was inserted into the XbaI-NdeI sites as a fragmentobtained after annealing the oligonucleotides:

4A (SEQ ID NO: 9) (5′ CTAGTGCCGGACCCGGTTCCAAGGCCGGCCGCAAGGCTGCCAGAACTGAGGAAGCACA) and 4B (SEQ ID NO: 10)(5′ TATGTGCTTCCTCAGTTCTGGCAGCCTTGCGGCCGGCCTTGGAACC GGGTCCGGCA).The resulting plasmid is pMSX₁₁AB₅kan2. The AvrII fragment, containingTp₁₁AB₅kan2 was cloned into pUTHg cut with AvrII and used forintegration into the genome of MBX379 and MBX245.

Plasmid pMSXTp₁₂AB₅kan2 was constructed as pMSXTP₁₁AB₅kan2 with thedistinction that the following oligonucleotides were used instead ofoligonucleotides 1A and 1B:

2A: (5′ TCCCCTGTCATAAAGTTGTCACTGCA) (SEQ ID NO: 11) and 2B(5′ GTGACAACTTTATGACAG GGGATGCA). (SEQ ID NO: 12).These oligonucleotides provide a consensus E. coli pho box and −35promoter region to generate a promoter that is potentially regulated bythe phosphate concentration in the medium.

pMSXTp₁₃AB₅kan2 was constructed to provide expression of AB₅ from apromoter that has been shown to be expressed under general stressconditions such as nutrient limitation, pH or heat shock, andadministration of toxic chemicals. The promoter region of uspA wasamplified using oligonucleotides UspUp

(5′ TGACCAACATACGA GCGGC) (SEQ ID NO: 13) and UspDwn (5′CTACCAGAACTTTGCTTTCC) (SEQ ID NO: 14)

in a PCR reaction consisting of an incubation at 95° C. for 3 min.followed by 30 cycles of 40 s at 95° C., 40 s at 42° C., an incubationfor 7 min. at 68° C., and final storage at 4° C. The approximately 350by PCR product was cloned into pCR2.1 (Invitrogen Corp., USA) togenerate pMBXp₁₃. An approximately 190 by HincII-MscI fragmentcontaining the promoter and transcriptional start site for uspA and thefirst 93 by of the uspA mRNA was cloned into blunt ended BamHI-Sse8387IpMSXTp₁kan2 to give pMSXTp₁₃kan2. Plasmid pMSXTp₁₃kan2 was then KpnIdigested, blunt ended with T4 polymerase and dephosphorylated using calfintestinal phosphatase. The AB₅ genes were isolated as a 2.0 kbEcoRI/Sse8387I fragment from pMSXAB₅, blunt ended using Klenow and T4polymerase and ligated into the KpnI site of pMSXTp₁₃kan2. In theresulting plasmid pMSXTp₁₃AB₅kan2, the phbAB and kan genes are expressedfrom the uspA (p₁₃) promoter.

The p_(n)AB₅kan (n=11, 12, 13) expression cassettes were then excised as2.8 kb AvrII fragments and ligated into the AvrII site of pUTHg andtransformed into E. coli strain CC118 λpir to obtain plasmidpMUXp_(n)AB₅kan. This plasmid was then transformed into E. coliS17-1λpir and used to insert p₁₁AB₅kan, p₁₂AB₅kan, and p₁₃AB₅kanexpression cassettes into the chromosome of E. coli strains byconjugation.

Example 6 Integration of C₅cat into the Chromosome of E. coli

C₅cat was introduced into the chromosome of MBX23 by conjugation usingS17-1 λpir (pMUXC₅cat) as the donor strain. The conjugation mixture wasspread on LB/Nl/Cm plates and integrants were obtained, 40% of whichwere sensitive to ampicillin, indicating that no plasmid was present inthese strains. Five integrants were transformed with pMSXAB₅cat (Ap^(r))and grown on LB/Ap/Cm/2% glucose to examine biosynthetic activity of PHBpolymerase (Table 4).

TABLE 4 Integrated Strains strain containing strain after strainpMSXAB5cat PHB phenotype plasmid curing MBX300 MBX305 ++++ MBX325 MBX301MBX308 +++ MBX331 MBX302 MBX310 ++++ MBX326 MBX303 MBX315 ++++ MBX327MBX304 MBX316 + MBX337

Example 7 Amplification of C5 Expression in Integrated Strains

Expression of PHB polymerase was increased by restreaking MBX326successively on LB plates containing 100, 200, 500, and 1000 μg/mlchloroamphenicol. Strain MBX379 was derived from MBX326 and exhibitedchloramphenicol resistance up to 1000 μg/ml. In Southern blot analysisof chromosomal DNA isolated from MBX379 and its predecessors, the phbC5copy-number had not increased. Western blot analysis indicated a strongincrease in PHB polymerase levels in cell free extracts of these strainswhen the phbAB genes were present on a plasmid.

Example 8 Integration of p₁₁AB₅kan, p₁₂AB₅kan and p₁₃AB₅kan into MBX379

S17-1 λpir strains with either pMUXp₁₁AB₅kan, pMUXp₁₂AB₅kan, orpMUXp₁₃AB₅kan were mated with MBX379. Transgenic strains in whichphbAB₅kan had integrated on the chromosome were selected on LB/Nl/Kmplates. Among the integrants, PHB producers were identified onLB/glucose plates. Representatives of the individual constructs wereMBX612 (MBX379::p₁₁AB₅kan), MBX677 (MBX379::p₁₂AB₅kan), and MBX680(MBX379::p₁₃AB₅kan). Southern blots and Western blots showed that thephbAB genes had integrated in the chromosome and were expressed in thesestrains as well. Table 5 shows the PHB accumulation levels of transgenicE. coli PHB producers grown in Luria-Bertani medium with 2% glucose orminimal E2 medium with 2% glucose and 0.5% corn steep liquor.

TABLE 5 PHB Accumulation Levels for Transgenic E. coli PHB Producers %PHB of cell dry weight strain LB/glucose E2 glucose MBX612 56 35 MBX67758 38 MBX680 39 50

Example 9 Selection and Bacteriophage P1 Transduction to Yield ImprovedStrains

The growth characteristics of MBX612, 677, and 680 were improved bybacteriophage P1 transduction. A single transduction step was requiredto transduce the C₅cat and AB₅kan alleles from the different strainsinto LS5218, indicating that the two separate integration cassettes werelocated close to each other on the chromosome. The resulting strains areMBX690 (from MBX681), MBX691 (from MBX677), and MBX698 (from MBX680).Repeated inoculation of MBX612 on minimal E2 medium with limitingnitrogen resulted in MBX681. Unlike the strains generated by P1transduction, MBX681 did not exhibit improved growth characteristics.Southern blots and Western blots show that phbC and the phbAB genes weresuccessfully transduced and were expressed in these strains as well.Table 6 below shows PHB accumulation levels for these transgenic E. coliPHB producers grown in Luria-Bertani medium with 2% glucose or minimalE2 medium with 2% glucose and 0.5% corn steep liquor.

TABLE 6 PHB Accumulation Levels for Transgenic E. coli PHB Producers %PHB of cell dry weight strain LB/glucose E2 glucose MBX681 54 22 MBX69052 44 MBX691 54 28 MBX698 37 15

Example 10 Further Improvements of Transgenic E. coli Strains for PHBProduction

Mutagenesis using NTG or EMS was used to further improve PHB productionin MBX680. Strains MBX769 and MBX777 were selected after treatment ofMBX680 with EMS and NTG, respectively. These strains were found to beable to grow on R2-medium supplied with 1% glucose, 0.5% corn steepliquor, and 1 mg/ml chloroamphenicol. MBX769 was grown in 50 ml R-10medium/0.5% CSL with 2 or 3% glucose at 37° C. for 20 to 26 hours. PHBwas accumulated to 71% of the cell dry weight. Similarly, MBX769 wasgrown in 50 ml LB with or without 0.375 g/L KH₂PO₄, 0.875 K₂HPO₄, 0.25(NH₄)₂SO₄, and a total of 50 g/L glucose (five aliquots were added overthe course of the incubation). After 63 hours of incubation, PHB hadaccumulated up to 96% of the cell dry weight.

The phbC and phbAB alleles from MBX777 were subsequently transduced intoLS5218, resulting in MBX820. Southern blots and Western blots show thatphbC and the phbAB genes were successfully transduced and were expressedin these strains as well. Table 7 shows the PHB accumulation levels ofthese transgenic E. coli PHB producers grown in Luria-Bertani mediumwith 2% glucose or minimal E2 medium with 2% glucose and 0.5% corn steepliquor.

TABLE 7 PHB Accumulation Levels for Transgenic E. coli PHB Producers %PHB of cell dry weight strain LB/glucose E2 glucose MBX680 39 50 MBX77767 57 MBX820 53 50

Example 11 Growth Characteristics of Transgenic E. coli PHB Producers

The introduction of phb genes into MBX245 (t_(d)=47 min.) wasaccompanied by a reduction in growth rate (MBX680, t_(d)=71 min.).Improved PHB production was achieved by EMS mutagenesis, but did notimprove the growth rate (MBX777, t_(d)=72 min.). P1 transduction of thePHB genes into a wild-type strain (MBX184) resulted in the same highgrowth rate as exhibited by MBX245 and PHB accumulation up to 50% of thecell dry weight in less than 24 hours (MBX820, t_(d)=45 min.).

Example 12 Plasmids for Chromosomal Integration of Other pha Genes

The integration of phbC, phbA, and phbB from Z. ramigera describedherein also is applicable to other pha genes, such as genes encoding PHBpolymerase from R. eutropha (C1), PHA polymerase from P. oleovorans(C3), PHB polymerase from A. caviae (C12), ACP::CoA transacylase from P.putida (G3), (R)-specific enouyl-CoA hydratase from A. caviae (J12), abroad substrate specific 3-ketoacyl-CoA thiolase from R. eutropha(A1-II), or a phasin from R. eutropha (P1-I and P1-II). These genes wereobtained by polymerase chain reaction amplification using the followingprimers:

C1 up (SEQ ID NO: 22) 5′ g-GAATTC-aggaggtttt-ATGGCGACCGGCAAAGGCGCGGCA G3′, C1 dw (SEQ ID NO: 23) 5′ GC-TCTAGA-AGCTT-tcatgccttggctttgacgtatcgc3′, C3 up (SEQ ID NO: 24)5′ g-GAATTC-aggaggtttt-ATGAGTAACAAGAACAACGATGAG C 3′, C3 dw (SEQ ID NO:25) 5′ GC-TCTAGA-AGCTT-tcaacgctcgtgaacgtaggtgccc 3′, C12 up (SEQ ID NO:26) 5′ g-GAATTC-aggaggtttt-ATGAGCCAACCATCTTATGGCCCG C 3′, C12 dw (SEQ IDNO: 27) 5′ GC-TCTAGA-AGCTT-TCATGCGGCGTCCTCCTCTGTTGGG 3′, G3 up (SEQ IDNO: 28) 5′ g-GAATTC-aggaggtttt-ATGAGGCCAGAAATCGCTGTACTT G 3′, G3 dw (SEQID NO: 29) 5′ GC-TCTAGA-AGCTT-tcagatggcaaatgcatgctgcccc 3′, J12 up (SEQID NO: 30) 5′ ag-GAGCTC-aggaggtttt-ATGAGCGCACAATCCCTGGAAGTA G 3′, J12 dw(SEQ ID NO: 31) 5′ GC-TCTAGA-AGCTT-ttaaggcagcttgaccacggcttcc 3′, A1-IIup (SEQ ID NO: 32) 5′ g-GAATTC-aggaggtttt-ATGACGCGTGAAGTGGTAGTGGTAA G3′, A1-II dw (SEQ ID NO: 33) 5′ GC-TCTAGA-AGCTT-tcagatacgctcgaagatggcggc3′, P1-I up (SEQ ID NO: 34)5′ g-GAATTC-aggaggtttt-ATGATCCTCACCCCGGAACAAGTT G 3′, P1-I dw (SEQ IDNO: 35) 5′ GC-TCTAGA-AGCTT-tcagggcactaccttcatcgttggc 3′, P1-II up (SEQID NO: 34) 5′ g-GAATTC-aggaggtttt-ATGATCCTCACCCCGGAACAAGTT G 3′, P 1-IIdw (SEQ ID NO: 36) 5′ GC-TCTAGA-AGCTT-tcaggcagccgtcgtcttctttgcc 3′.PCR reactions included 10 pmol of each primer, 1 to 5 μl of chromosomalDNA or boiled cells, and 45 μl PCR mix from Gibco BRL (Gaithersburg,Md.). Amplification was by 30 cycles of 60 s incubation at 94° C., 60 sincubation at a temperature between 45° C. and 68° C. and 1 to 3 minutesincubation at 72° C. PCR products were purified, digested with EcoRI andHindIII, blunt ended with the Klenow fragment of DNA polymerase, andcloned in the SmaI site of pMSXcat, pMSXkan, pMNXcat, or pMNXkanaccording to the schemes shown in FIGS. 1A-1C, 2A and 2B, and 3A-3C.pMUXpha was derived from pUTHg or pUTkan; and pMLXpha was derived frompLOFHg, where pha stands for the pha gene of choice. These plasmids wereused for integration of the desired pha gene into the chromosome of E.coli or any other Gram-negative microbial strain suitable for PHAproduction

Example 13 PHBV Copolymer Producing Transgenic E. coli Strains

E. coli strains with chromosomally integrated phb genes such asdescribed above also can be used to produce PHBV copolymers. PHBV isgenerally synthesized in fermentation systems where propionic acid isco-fed with glucose or other carbohydrate. After uptake, propionate isconverted to propionyl-CoA, which by the action of acyl-CoA thiolase and3-ketoacyl-CoA reductase is converted to 3-hydroxyvaleryl-CoA (3HV-CoA).3HV-CoA is subsequently polymerized by PHA polymerase.

The capacity to accumulate PHBV can be increased by increasing levels ofenzymes that specifically synthesize HV monomers. Such enzymes may beinvolved in the uptake of propionic acid, in the activation of propionicacid to propionyl-CoA or in any of the PHB biosynthetic enzymes.Additionally, alternative enzymes can be isolated from other sources, orpropionyl-CoA can be obtained from alternative pathways, e.g. from themethylmalonyl-CoA pathway. In this pathway, succinyl-CoA is converted tomethylmalonyl-CoA which is then decarboxylated to yield propionyl-CoA.

Example 14 PHB-4HB Copolymer Producing Transgenic E. coli Strains

Homopolymers and copolymers containing 4HB monomers can be produced bytransgenic E. coli strains. Incorporation of 4HB from 4HB-CoA can beachieved by feeding 4-hydroxybutyrate to the PHA producing organisms.4HB is activated to 4HB-CoA either through a 4-hydroxybutyryl-CoAtransferase such as hbcT (OrfZ) from Clostridium kluyveri or by anendogenous E. coli enzyme or by any other enzyme with this capability. AP4HB homopolymer is produced when the transgenic E. coli strain containsonly the phbC gene. 4HB containing copolymers can be synthesized whenthe transgenic E. coli strain contains genes encoding the complete PHBbiosynthetic pathway.

E. coli MBX821 (LS5218::C₅-cat³⁷⁹, atoC^(c)) was grown in Luria-Bertanimedium and resuspended in 100 ml 10% LB with 5 g/L 4HB and 2 g/Lglucose. After incubation of this culture for 24 hours, PHA wascharacterized and identified as containing only 4HB monomers. Similarly,E. coli MBX777 with a plasmid containing hbcT such as pFS16, was grownin LB/4HB (5 g/L) and the resulting polymer was identified as PHB4HBwith 35.5% 4HB monomers.

Example 15 Production of poly(4-hydroxybutyrate) from 4-hydroxybutyratein Recombinant E. coli with No Extrachromosomal DNA

Poly(4-hydroxybutyrate) can be synthesized from 4-hydroxybutyrate by E.coli expressing 4-hydroxybutyryl-CoA transferase (hbcT) and PHA synthase(phaC) genes from a plasmid. If these genes are integrated into the E.coli chromosome and expressed at high levels, the recombinant E. colishould be able to synthesize poly(4-hydroxybutyrate) from4-hydroxybutyrate. The hbcT and phbC genes were inserted into pUTHg(Herrero, et al., J. Bacteriol. 172:6557-67, 1990) as follows. pMSXC₅catand pFS16 were both digested with BamHI and SalI. The large fragment ofpMSXC₅cat and the fragment of pFS16 containing the hbcT gene thusobtained were ligated together using T4 DNA ligase to formpMSXC₅hbcT-cat. The fragment containing the phaC, hbcT, and cat geneswas removed from pMSXC₅hbcT-cat by digestion with AvrII, and it wasinserted using T4 DNA ligase into pUTHg that had been digested withAvrII and treated with calf intestinal alkaline phosphatase to preventself-ligation. The plasmid thus obtained was denoted pMUXC₅hbcT-cat. Theplasmid pMUXC₅hbcT-cat was replicated in MBX129 and conjugated intoMBX1177. The strain MBX1177 is a spontaneous mutant of E. coli strainDH5α that was selected for its ability to grow on minimal4-hydroxybutyrate agar plates. MBX1177 is also naturally resistant tonalidixic acid. The recipient cells were separated from the donor cellsby plating on LB-agar supplemented with 25 μg/mL chloramphenicol and 30μg/mL nalidixic acid. Survivors from this plate were restreaked onminimal medium, containing, per liter: 15 g agar; 2.5 g/L LB powder(Difco; Detroit, Mich.); 5 g glucose; 10 g 4-hydroxybutyrate; 1 mmolMgSO₄; 10 mg thiamine; 0.23 g proline; 25.5 mmol Na₂HPO₄; 33.3 mmolK₂HPO₄; 27.2 mmol KH₂PO₄; 2.78 mg FeSO₄.7H₂O; 1.98 mg MnCl₂.4H₂O; 2.81mg CoSO₄.7H₂O; 0.17 mg CuCl₂.2H₂O; 1.67 mg CaCl₂.2H₂O; 0.29 mgZnSO₄.7H₂O; and 0.5 mg chloramphenicol. Colonies from this plate thatappeared to be especially white and opaque were evaluated in shakeflasks containing the same medium as above except without agar. Theindividual colonies were first grown in 3 mL of LB medium for 8 hours,and 0.5 mL of each culture was used to inoculate 50 mL of the mediumdescribed above. These flasks were incubated at 30° C. for 96 hours. Oneisolate was found by GC analysis (for which the cells were removed fromthe medium by centrifugation for 10 minutes at 2000×g, washed once withwater and centrifuged again, then lyophilized) to contain 4.9%poly(4-hydroxybutate) by weight. This strain was denoted MBX1462 andselected for further manipulations. MBX1462 was treated with the mutagen1-methyl-3-nitro-1-nitrosoguanidine (MNNG), a chemical mutagen, byexposing a liquid culture of MBX1462 to 0.1 mg/mL MNNG for 90 minutes.It was found that 99.8% of the cells were killed by this treatment. Theplating and shake flask experiment described above was repeated, and oneisolate was found by GC analysis to contain 11% poly(4-hydroxybutate) byweight. This strain was denoted MBX1476 and selected for furthermanipulations. The NTG treatment was repeated and killed 96.3% of thecells. The plating and shake flask experiment described above wasrepeated once again, and one isolate was found by GC analysis to contain19% poly(4-hydroxybutate) by weight. This strain was denoted MBX1509.

Example 15 PHBH Copolymer Producing Transgenic E. coli Strains

E. coli MBX240 is an XL1-blue (Stratagene, San Diego, Calif.) derivativewith a chromosomally integrated copy of the PHB polymerase encoding phbCgene from Ralstonia eutropha. This strain does not form PHAs from carbonsources such as glucose or fatty acids, because of the absence ofenzymes converting acetyl-CoA (generated from carbohydrates such asglucose) or fatty acid oxidation intermediates, into(R)-3-hydroxyacyl-CoA monomers for polymerization. pMSXJ12 wasconstructed by inserting the phaJ gene from A. caviae digested withEcoRI and PstI into the corresponding sites of pUC18Sfi. The phaJ genewas obtained by polymerase chain reaction using the primers Ac3-5′:

5′ AGAATTCAGGAGGACGCCGCATGAGCGCACAATCCCTGG (SEQ ID NO: 37) and Ac3-3′:5′ TTCCTGCAGCTCAAGGCAGCTTGACCACG (SEQ ID NO: 38)

using a PCR program including 30 cycles of 45 s at 95° C., 45 s at 55°C. and 2.5 minutes at 72° C. Transformants of E. coli MBX240 withplasmid pMTXJ12 containing the (R)-specific enoyl-CoA hydratase encodedby the phaJ gene from Aeromonas caviae were grown on Luria-Bertanimedium with 10 mM octanoate and 1 mM oleate. After 48 hours of growth,cells were harvested from a 50 ml culture by centrifugation and the cellpellet lyophilized. Lyophilized cells were extracted with chloroform (8ml) for 16 hours and PHA was specifically precipitated from thechloroform solution by adding the chloroform layer to a 10-fold excessethanol. Precipitation was allowed to occur at 4° C. and the solidpolymer was air dried and analyzed for composition by acidicbutanolysis. Butylated PHA monomers were separated by gas chromatographyand identified the PHA as apoly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer with 2.6%3-hydroxyhexanoate monomers.

Example 16 Construction of Transgenic E. coli Strains for Screening ofNew and/or Improved Genes for PHA Production

The phbC gene was introduced into an E. coli cloning strain bybacteriophage P1 transduction. In a procedure similar to that followedfor phbC₅ integration, the phbC gene from R. eutropha was integratedinto the chromosome of MBX23, resulting in MBX143. After chloramphenicolamplification, MBX150, which is resistant to 500 μg/ml chloramphenicol,was isolated. A bacteriophage P1 lysate grown on MBX150 was used totransduce the phbC-cat allele into XL1-Blue [pT7-RecA]. Plasmid pT7RecAexpresses a functional RecA protein which is required for successful P1transduction. The resulting strain MBX240 is an XL1-Blue derivative witha functional PHB polymerase expressed from the chromosome. MBX613 andMBX683 were developed using the same procedures. These strains werederived from MBX245 and XL1-Blue, respectively, and contain integratedAB₅cat (MBX613) or p₁₃AB₅kan (MBX683) operons.

Example 17 Identification of Genes Encoding New, Improved, or AncillaryPHA Biosynthetic Enzymes

MBX240, 613, and 683 are three strains that can be used in screeningprocedures for new or improved PHA genes. Using these strains, thefollowing genes have been identified: phbCABFA2 from P. acidophila andphbCAB from A. latus. In addition, the phaJ gene from A. caviae wasfunctionally expressed in MBX240 to produce PHA from fatty acids.Besides PHA biosynthetic genes specific for C₃ to C₆ monomers, PHAbiosynthetic enzymes for PHAs consisting of medium side-chain 3-hydroxyacids can also be expressed in E. coli. Such strains are useful inidentifying additional PHA biosynthetic enzymes.

Example 18 Integration of pha Genes in R. eutropha

The plasmids described in the previous examples were used to integratepha genes in R. eutropha. Using a PHA-negative mutant of R. eutrophasuch as #2 (Peoples & Sinskey, J. Biol. Chem. 264:15298-303 (1989)) orPHB⁻4 (Schubert, et al., J. Bacterial. 170:5837-47 (1988)), PHAformation was restored by integration of phaC from A. caviae incombination with phbAB from Z. ramigera or phaJ from A. caviae. Theresulting strains produced PHAs to variable levels, with a molecularweight in the range of 400,000 to 10,000,000 Da and with a compositionthat includes monomers such as 3-hydroxyhexanoate and3-hydroxyoctanoate.

Example 19 Integration of pha Genes in P. putida

The plasmids described in the previous examples were used to integratepha genes into Pseudomonas putida. The PHA-negative phenotype of P.putida GPp104 (Huisman et al., J. Biol. Chem. 266:2191-98 (1991)) wasrestored by integration of a phaC3kan cassette where phaC3 encodes thePHA polymerase from P. oleovorans. Integration of phaC3kan usingpMUXC3kan was also applied to generate mutants of P. putida withmutations in genes encoding enzymes that affect PHA metabolism otherthan phaC. The PHA polymerase gene from A. caviae was also introduced into the chromosome to result in a strain that produces PHAs including3-hydroxy fatty acids in the C₃ to C₉ range.

Example 20 Chromosomal Integration of phaC Genes to Control MolecularWeight of the Resulting PHA

It is well known that the concentration of PHA polymerase determines themolecular weight of the produced PHA when substrate is available inexcess. Variation of the molecular weight is desirable as polymerproperties are dependent on molecular weight. Chromosomal integration ofphb genes results in variable levels of expression of the pha gene asdetermined by the chromosomal integration site. It is therefore possibleto obtain different transgenic bacteria that have variable levels ofphaC expression and hence produce PHAs of variable molecular weight.With this system, it is possible to produce PHAs with molecular weightsof greater than 400,000 Da and frequently even, in excess of 1,000,000Da. This procedure is applicable to any gram-negative bacterium in whichthe pUT or pLOF derived plasmids can be introduced, such as E. coli, R.eutropha, P. putida, Klebsiella pneumoniae, Alcaligenes latus,Azotobacter vinelandii, Burkholderia cepacia, Paracoccus denitrificansand in general in species of the Escherichia, Pseudomonas, Ralstonia,Burkholderia, Alcaligenes, Klebsiella, Azotobacter genera.

Example 21 Integration of the PHB Genes as a Single Operon

A plasmid, pMSXABC₅kan, was constructed such that the thiolase (phbA),reductase (phbB), and PHB synthase (phbC) genes from Zoogloea ramigeraand the kanamycin resistance gene (kan) were linked as an operon in thevector pUC18Sfi. This expression cassette was then excised as an AvrIIfragment and inserted into the AvrII site of pUT to obtain pMUXABC₅kan.

S17-1 λpir strains with pMUXABC₅kan were mated with MBX247. Transgenicstrains in which phbABC₅kan had integrated into the chromosome wereselected on LB/Nl/Km plates. Among the integrants, PHB producers wereidentified on LB/glucose plates. One strain thus constructed, MBX1164,was selected for further study.

Thiolase (Nishimura et al., 1978, Arch. Microbiol. 116:21-24) andreductase (Saito et al., 1977, Arch. Microbiol. 114:211-217) assays wereconducted on MBX1164 crude extracts. The cultures were grown in 50 mL of0.5×E2 medium supplemented with 20 g/L glucose. One unit (U) was definedas the amount of enzyme that converted 1 μmol of substrate to productper min. 3-Ketothiolase activity was determined to be 2.23±0.38 and2.48±0.50 U/mg in two independent trials, and 3-hydroxybutyryl-CoAreductase activity was determined to be 4.10±1.51 and 3.87±0.15 U/mg intwo independent trials.

Strain MBX1164 was evaluated for its PHB-producing ability in squareshake bottles. The cells were grown in 2 mL of LB, and 0.1 mL of thiswas used as an inoculum for the 50-mL shake bottle culture. The shakebottle contained E2 medium supplemented with 0.25% corn steep liquor(Sigma, St. Louis, Mo.) and 20 g/L glucose. After incubation at 30° C.for 48 hours with shaking at 200 rpm, the biomass concentration hadreached 2.6 g/L, and the PHB concentration had reached 11.7 g/L; thusthe cells contained 82% PHB by weight.

Example 22 Integration of the Pseudomonas oleovorans PHA Synthase intothe E. coli Chromosome

A PHA synthase (phaC) cassette from the P. oleovorans chromosome and apromoterless chloramphenicol resistance gene were inserted into pUC118such that an operon of the two genes was formed; i.e., they wereoriented in the same direction and could be transcribed on the samemRNA. The sequence of the P. oleovorans phaC gene is shown below. ThephaC-cat operon was excised from this plasmid by digestion with KpnI andHindIII and ligated to pUC18SfiI that had been digested with the sametwo enzymes to form pMSXC₃cat. This allowed the phaC-cat operon to beflanked by AvrII sites. The phaC-cat operon was removed from pMSXC₃catby digestion with AvrII and FspI. Because the two AvrII fragments ofpMSXC₃cat were nearly the same size, FspI was used to facilitateisolation of the phaC-cat operon by cutting the rest of the vector intotwo pieces. The AvrII fragment was ligated to pUTkan which had beendigested with AvrII and treated with alkaline phosphatase to preventself-ligation. The plasmid thus produced was denoted pMUXC₃cat. Theoperon on this plasmid actually consisted of phaC-cat-kan. Strain CC118λpir (a λpir lysogenic strain) was transformed with pMUXC₃cat to producestrain MBX130. Equal amounts of strains MBX130 and MBX245 were mixed onan LB agar plate and incubated for 8 hours at 37° C. The mixed cellswere then used as an inoculum for an overnight 37° C. culture ofLB-chloramphenicol (25 μg/mL)-nalidixic acid (30 μg/mL). Single colonieswere isolated from this culture by plating on LB-chloramphenicol (25μg/mL)-nalidixic acid (30 μg/mL)-kanamycin (25 μg/mL). The colonies thusisolated have a transducible phaC-cat-kan cassette on the chromosome, asshown by the ability to use P1 transduction to introduce the cassetteinto the chromosome of other strains and select for resistance to bothchloramphenicol and kanamycin.

Pseudomonas oleovorans PHA Synthase (phaC).

(SEQ ID NO: 39) ATGAGTAACAAGAACAACGATGAGCTGCAGCGGCAGGCCTCGGAAAACACCCTGGGGCTGAACCCGGTCATCGGTATCCGCCGCAAAGACCTGTTGAGCTCGGCACGCACCGTGCTGCGCCAGGCCGTGCGCCAACCGCTGCACAGCGCCAAGCATGTGGCCCACTTTGGCCTGGAGCTGAAGAACGTGCTGCTGGGCAAGTCCAGCCTTGCCCCGGAAAGCGACGACCGTCGCTTCAATGACCCGGCATGGAGCAACAACCCACTTTACCGCCGCTACCTGCAAACCTATCTGGCCTGGCGCAAGGAGCTGCAGGACTGGATCGGCAACAGCGACCTGTCGCCCCAGGACATCAGCCGCGGCCAGTTCGTCATCAACCTGATGACCGAAGCCATGGCTCCGACCAACACCCTGTCCAACCCGGCAGCAGTCAAACGCTTCTTCGAAACCGGCGGCAAGAGCCTGCTCGATGGCCTGTCCAACCTGGCCAAGGACCTGGTCAACAACGGTGGCATGCCCAGCCAGGTGAACATGGACGCCTTCGAGGTGGGCAAGAACCTGGGCACCAGTGAAGGCGCCGTGGTGTACCGCAACGATGTGCTGGAGCTGATCCAGTACAACCCCATCACCGAGCAGGTGCATGCCCGCCCGCTGCTGGTGGTGCCGCCGCAGATCAACAAGTTCTACGTATTCGACCTGAGCCCGGAAAAGAGCCTGGCACGCTACTGCCTGCGCTCGCAGCAGCAGACCTTCATCATCAGCTGGCGCAACCCGACCAAAGCCCAGCGCGAATGGGGCCTGTCCACCTACATCGACGCGCTCAAGGAGGCGGTCGACGCGGTGCTGGCGATTACCGGCAGCAAGGACCTGAACATGCTCGGTGCCTGCTCCGGCGGCATCACCTGCACGGCATTGGTCGGCCACTATGCCGCCCTCGGCGAAAACAAGGTCAATGCCCTGACCCTGCTGGTCAGCGTGCTGGACACCACCATGGACAACCAGGTCGCCCTGTTCGTCGACGAGCAGACTTTGGAGGCCGCCAAGCGCCACTCCTACCAGGCCGGTGTGCTCGAAGGCAGCGAGATGGCCAAGGTGTTCGCCTGGATGCGCCCCAACGACCTGATCTGGAACTACTGGGTCAACAACTACCTGCTCGGCAACGAGCCGCCGGTGTTCGACATCCTGTTCTGGAACAACGACACCACGCGCCTGCCGGCCGCCTTCCACGGCGACCTGATCGAAATGTTCAAGAGCAACCCGCTGACCCGCCCGGACGCCCTGGAGGTTTGCGGCACTCCGATCGACCTGAAACAGGTCAAATGCGACATCTACAGCCTTGCCGGCACCAACGACCACATCACCCCGTGGCAGTCATGCTACCGCTCGGCGCACCTGTTCGGCGGCAAGATCGAGTTCGTGCTGTCCAACAGCGGCCACATCCAGAGCATCCTCAACCCGCCAGGCAACCCCAAGGCGCGCTTCATGACCGGTGCCGATCGCCCGGGTGACCCGGTGGCCTGGCAGGAAAACGCCACCAAGCATGCCGACTCCTGGTGGCTGCACTGGCAAAGCTGGCTGGGCGAGCGTGCCGGCGAGCTGAAAAAGGCGCCGACCCGCCTGGGCAACCGTGCCTATGCCGCTGGCGAGGCATCCCCGGGCACCTACGTTCACGAGCGTTGA

Modifications and variations of the present invention will be obvious tothose of skill in the art from the foregoing detailed description. Suchmodifications and variations are intended to come within the scope ofthe following claims.

1. A genetically engineered microorganism having at least one geneinvolved in synthesis of polyhydroxyalkanoates selected from the groupconsisting of thiolase, reductase, PHB synthase, PHA synthase, acyl-CoAtransferase, enoyl-CoA hydratase, integrated into the chromosome, whichproduce polyhydroxyalkanoate.
 2. The microorganism of claim 1 selectedfrom the group consisting of E. coli, Alcaligenes latus, Alcaligeneseeutrophus, Azotobacter, Pseudomonas putida, and Ralstonia eutropha. 3.The microorganism of claim 1 wherein the gene is inserted usingtransposan mutagenesis.
 4. The microorganism of claim 1 comprisingmultiple genes involved in synthesis of polyhydroxyalkanoate wherein thegenes are integrated operably linked as an operon.
 5. The microorganismof claim 1 wherein the gene is integrated operably linked under thecontrol of a promoter.
 6. The microorganism of claim 1 wherein the geneis integrated operably linked with upstream activating sequences
 7. Themicroorganism of claim 1 wherein the gene is integrated operably linkedwith mRNA stabilizing sequences.
 8. The microorganism of claim 5 whereinthe gene is operably linked with promoter including a consensus E. colipho box and −35 promoter region that is regulated by the phosphateconcentration in the medium.
 9. The microorganism of claim 5 wherein thepromoter is selected from the group consisting of promoter that inducesexpression under general stress conditions such as nutrient limitation,pH or heat shock, and administration of toxic chemicals.
 10. Themicroorganism of claim 1 wherein the gene is integrated operably linkedwith a selection marker.
 11. The microorganism of claim 1 wherein thegene is isolated or derived from a microorganism selected from the groupconsisting of A. eutrophus, Aeromonas caviae, Zoogloea ramigera,Nocardia, Rhodococcus, Pseudomonas Sp. 61-3, Pseudomonas acidophila,Pseudomonas oleovarans, Chromobacterium violaceum, and Alcaligeneslatus.
 12. The microorganism of claim 1 wherein the gene is selectedfrom the group consisting of PHB polymerase from R. eutropha (C1), PHApolymerase from P. oleovorans (C3), PHB polymerase from A. caviae (C12),ACP::CoA transacylase from P. putida (G3), (R)-specific enouyl-CoAhydratase from A. caviae (J12), a broad substrate specific3-ketoacyl-CoA thiolase from R. eutropha (A1-II), and phasins from R.eutropha (P1-I and P1-II).
 13. The microorganism of claim 1 wherein thegene is integrated as a single copy on the chromosome of themicroorganism.
 14. A method for screening for a gene involved insynthesis of polyhydroxyalkanoates that enhances production comprisingmutating a genetically engineered microorganism having at least one geneinvolved in synthesis of polyhydroxyalkanoates selected from the groupconsisting of thiolase, reductase, PHB synthase, PHA synthase, acyl-CoAtransferase, enoyl-CoA hydratase, integrated into the chromosome, whichproduces polyhydroxyalkanoate, and screening for enhanced production ofpolyhydroxyalkanoates.
 15. The method of claim 14 wherein the gene isisolated or derived from a microorganism selected from the groupconsisting of A. eutrophus, Aeromonas caviae, Zoogloea ramigera,Nocardia, Rhodococcus, Pseudomonas Sp. 61-3, Pseudomonas acidophila,Pseudomonas oleovarans, Chromobacterium violaceum, and Alcaligeneslatus.
 16. The method of claim 14 wherein the microorganisms are missingone or more genes required for production of polyhydroxyalkanoates. 17.The method of claim 14 wherein the microorganisms producepolyhydroxyalkanoates, further comprising selecting genes which resultin increased polyhydroxyalkanoate production.
 18. A method for producingpolyhydroxyalkanoates comprising culturing genetically engineeredmicroorganisms having at least one gene involved in synthesis ofpolyhydroxyalkanoates selected from the group consisting of thiolase,reductase, PHB synthase, PHA synthase, acyl-CoA transferase, enoyl-CoAhydratase, integrated into the chromosome, with appropriate substrateunder conditions wherein the microorganisms producepolyhydroxyalkanoate.
 19. The method of claim 18 wherein themicroorganisms comprise multiple genes involved in synthesis ofpolyhydroxyalkanoate wherein the genes are integrated operably linked asan operon.
 20. The method of claim 18 wherein the microorganismscomprise genes selected from the group consisting of genes integratedoperably linked under the control of a promoter, genes integratedoperably linked with upstream activating sequences, genes integratedoperably linked with mRNA stabilizing sequences, genes operably linkedwith a promoter including a consensus E. coli pho box and −35 promoterregion that is regulated by the phosphate concentration in the medium,and genes integrated operably linked with a selection marker.